Diagnostic method for cancer characterized in the detection of the deletion of g-csf exon 3

ABSTRACT

Disclosed are a method, a composition, a microarray, an antibody and a kit for diagnosis and prognosis of cancer, based on detection of deletion of the exon 3 region of G-CSF gene or levels of a mutated G-CSF protein having a deletion of an amino acid sequence corresponding to the exon 3 region, wherein the deletion of the exon 3 region of the G-CSF gene is used as a cancer biomarker.

CONTINUING DATA

The present application is a divisional of U.S. patent application Ser.No. 11/059,222, filed Feb. 16, 2005 (pending), which is a divisional ofU.S. patent application Ser. No. 10/490,502, filed Mar. 22, 2004 (nowabandoned), which is a national phase of PCT Application No.PCT/KR/2002/01825, filed Sep. 28, 2002, which claims the benefit ofpriority to Korean application number 2001-0060826, filed Sep. 28, 2001,the contents of which are incorporated by reference herein in theirentirety.

TECHNICAL FIELD

The present invention relates to a method of diagnosing cancer based onmodified features in granulocyte colony stimulating factor (G-CSF) mRNAor protein. More particularly, the present invention relates to adiagnostic and prognostic method for cancer based on skipping of theexon 3 region of the G-CSF gene at mRNA or protein levels, whereinskipping of G-CSF exon 3 is used as a diagnostic cancer marker.

PRIOR ART

Cancer is a leading cause of death in developed nations. For thisreason, a major interest in cancer therapy is to develop methods forearly diagnosis and treatment of cancer. Typically, late-stage cancer isalmost incurable, whereas, at the early stage, cancer can be moreeffectively treated and therapeutic methods for early-stage cancer aresimpler. Therefore, there is an urgent need for development of methodsfor accurately and quickly diagnosing cancer.

At present, cancer diagnosis is generally achieved by morphologicalanalysis using microscopes such as an optical microscope or electronmicroscope, immunohistochemical assays which detect proteinsspecifically expressed in cancer tissues (Iran. Biomed. J. 3 (3 & 4):99-101, 1999; and Lancet 2:483-6, 1986), or molecular analysis ofabnormal biomolecules found in cancer tissues, such as mutated genes. Incomparison with the molecular diagnosis, the morphological andimmunohistochemical diagnosis requires much longer time and higher cost.Because of comprising a relatively simple procedure and yielding resultsin a short time, the molecular diagnosis methods are a focus fordeveloping novel diagnostic methods for cancer. Recently, a protein chipsystem for diagnosing various cancers has been developed by Health DigitInc. in Shanghai, China, and gained approval for clinical tests from theChinese State Drug Admistration (CSDA). Such an approval is the first inthe world (www.health-digit.com). However, the protein chip system doesnot use only a biomarker to diagnose all kinds of cancer, but uses 10 ormore proteins.

To effectively apply such diagnostic methods to cancer diagnosis, it ismost important to select cancer diagnostic markers capable of moreaccurately and easily discovering incidence of cancer. As diagnosticcancer markers, several genes (Steve M. et al., J. Clin. Oncology20:3165-3175, 2002; and Sridlhar R. et al, J. Clin. Oncology20:1932-1941, 2002) and proteins (Goessl et al., Urology 58:335-338,2001; Zhou et al., Breast Cancer Res Treat 66:217-224, 2001; and C K Kimet al., Korea Pat. Publication No. 2001-0061173) have been reported, andsome of them are being clinically used for diagnosis of cancer. Theconventional cancer biomarkers are unable to detect all kinds of cancer,as follows. The known cancer biomarkers which have low organspecificity, such as CEA, BFP, TPA and TAP, also, have low sensitivity,thus generating false positive data. Also, the biomarkers which havehigh organ specificity, exemplified by AFP, PIVKA II, Esterase I,CA19-9, CA50, Span-1 antigen, CA15-3 and BCA 225, are useful only fortarget organs. Therefore, for accurate, economical and simple diagnosisof cancer, there is an urgent need for development of new markerscapable of diagnosing a variety of cancers.

DISCLOSURE OF THE INVENTION

Leading to the present invention, the thorough and intensive researchinto a cancer biomaker capable of diagnosing a variety of cancers,conducted by the present inventors, resulted in the finding that exon 3skipping occurs during transcription of the G-CSF gene in cancerpatients, and use of G-CSF mRNA fragment or protein as a diagnosticcancer marker can achieve diagnosis of a variety of cancer, wherein thediagnosis is performed simply and quickly, as well as being economical.

In an aspect of the present invention, there is provided a mutated G-CSFmRNA fragment having a deletion of an exon 3 region for use as adiagnostic cancer marker.

In another aspect of the present invention, there is provided a mutatedG-CSF protein having a deletion of an amino acid sequence correspondingto an exon 3 region for use as a cancer diagnostic marker.

In a further aspect of the present invention, there is provided amicroarray or membrane for diagnosis of cancer comprising (a) a DNAfragment corresponding to exon 3 of a G-CSF gene, and (b) at least oneof DNA fragments corresponding to exons 1, 2, 4 and 5 of said G-CSFgene.

In a still further aspect of the present invention, there is provided adiagnostic agent for cancer comprising an antibody against a mutatedG-CSF protein having a deletion of an amino acid sequence correspondingto an exon 3 region of said G-CSF protein.

In a still further aspect of the present invention, there is provided adiagnostic kit for cancer comprising an antibody against a mutated G-CSFprotein having a deletion of an amino acid sequence corresponding to anexon 3 region of said G-CSF protein.

In a still further aspect of the present invention, there is provided amicroarray or membrane for diagnosis of cancer comprising an antibodyagainst a mutated G-CSF protein having a deletion of an amino acidsequence corresponding to the exon 3 region.

In a still further aspect of the present invention, there is provided adiagnostic method for cancer comprising the steps of: (a) obtaining aG-CSF nucleic acid sample from mammalian tissues or cells; (b)amplifying G-CSF region from the nucleic acid sample obtained; (c)detecting a deletion of exon 3 of said G-CSF gene in the amplifiedsample.

In a still further aspect of the present invention, there is provided adiagnostic method for cancer comprising the steps of: (a) obtaining aG-CSF protein sample from mammalian tissues or cells; and (b) detectinga deletion of an amino acid sequence corresponding to exon 3 of G-CSFgene in the G-CSF protein sample.

In a still further aspect of the present invention, there are providedprimers for use in amplification of G-CSF gene in a G-CSF nucleic acidsample obtained from mammalian tissues or cells.

BRIEF DESCRIPTION OF THE DRAWINGS

The above and other objects, features and other advantages of thepresent invention will be more clearly understood from the followingdetailed description taken in conjunction with the accompanyingdrawings, in which:

FIG. 1 is a process for normal transcription, splicing and translationof the human G-CSF gene;

FIG. 2 shows positions of primers used in PCR on a structural map ofhuman G-CSF gene comprising five exons;

FIG. 3A is a photograph showing PCR1 products separated on an agarosegel (M: size marker, lane 1: normal cell line, lane 2: YCC-7, lane 3:AGS, lane 4: SNU-1, lane 5: MDA-MB-231, lane 6: MCF-7, lane 7: SK-BR-3,lane 8: HT-1080, lane 9: HCT-116, and lane 10: COLO205);

FIG. 3B is a photograph showing PCR1 products separated on an agarosegel (M: size marker, lane 1: normal cell line, lane 2: DLD-1, lane 3:HT-29, lane 4: A549, lane 5: NCI-H460, lane 6: HeLa, lane 7: C-33A, lane8: B16, lane 9: U-87MG);

FIG. 4 shows a result of nucleotide sequence analysis of human G-CSFgene derived from normal cells;

FIG. 5 shows a result of nucleotide sequence analysis of human G-CSFgene derived from tumor cells;

FIG. 6A is a photograph showing PCR2 products separated on an agarosegel (each lane is the same cell line as in FIG. 3A);

FIG. 6B is a photograph showing PCR2 products separated on an agarosegel (each lane is the same cell line as in FIG. 3B);

FIG. 7A is a photograph showing PCR3 products separated on an agarosegel (each lane is the same cell line as in FIG. 3A);

FIG. 7B is a photograph showing PCR3 products separated on an agarosegel (each lane is the same cell line as in FIG. 3B);

FIG. 8 shows a result of hybridization of exon 2 DNA and exon 3 DNAbound to a nylon membrane with targets derived from tumor cells (A:YCC-7, B: AGC, C: HT-29, D: A549, E: MCF-7, and F: U-87MG);

FIG. 9 shows a result of analysis of deletion of exon 3 of G-CSF gene bya DNA chip;

FIG. 10 shows a result of SDS-PAGE of a purified recombinant mutatedG-CSF protein having a deletion of an amino acid sequence correspondingto the exon 3 region;

FIG. 11 is a graph showing levels of a mutated G-CSF protein having adeletion of an amino acid sequence corresponding to the exon 3 region innormal humans and cancer patients, which are measured using an antibodyto the mutated G-CSF protein;

FIG. 12 is a process for construction of plasmid p19CSF; and

FIG. 13 is a process for construction of recombinant plasmidpED-CSF4BLIIE expressing a mutated G-CSF protein having a deletion ofthe amino acid sequence corresponding to exon 3.

BEST MODES FOR CARRYING OUT THE INVENTION

The present invention is generally directed to a method for diagnosisand prognosis of cancer by analyzing the presence or absence of exon 3skipping of G-CSF gene at RNA or protein levels, wherein the G-CSF exon3 skipping is used as a diagnostic cancer marker.

Colony stimulating factor (CSF), produced in macrophages, T cells andfibroblasts, is widely distributed in a normal body. CSF is largelyclassified into granulocyte colony-stimulating factor (G-CSF),macrophage colony-stimulating factor (M-CSF) and granulocyte-macrophagecolony-stimulating factor (GM-CSF). Of them, G-CSF plays an importantrole in production of several blood cells during proliferation anddifferentiation of hemopoietic stem cells. The major role of G-CSF is toincrease the number of granulocytes, especially, neutrophils functioningto protect the body against foreign pathogens. The recently widely usedchemotherapy for proliferative tumor, which has the effect of inhibitinggrowth of tumor cells, is disadvantageous in terms of inhibitingdivision and maturation of neutrophil precursor cells, and thus reducingthe immunoprotective ability of patients in response to infection. Whenadministered to patients receiving such chemotherapy, G-CSF is known tobe effective in stimulating neutrophil proliferation and thus protectingand treating infectious diseases. In 1986, Nagata et al. reported thenucleotide sequence of human G-CSF gene and its expression in COS cells(Nagata et al., Nature 319:415-418, 1986).

Human G-CSF (hG-CSF) is a glycoprotein comprising a secretory signalpeptide consisting of 30 amino acids and 174 amino acids, and containsfive cystein residues. Four of the five cysteins, forming two disulfidebonds (Cys36-Cys42, Cys64-Cys74), play an important role in folding andbiological activity of hG-CSF protein (Hill et al., Proc. Natl. Acad.Sci. USA 90:5167-5171, 1993). With reference to FIG. 1, hG-CSF geneconsists of five exons and four introns. A mature hG-CSF mRNA isproduced by removing four introns from an hG-CSF mRNA transcripttranscribed from genomic DNA through splicing. The mature hG-CSF mRNA istranslated into an hG-CSF precursor protein consisting of 204 aminoacids, and a secretory signal peptide of 30 amino acids at theN-terminus is removed from the precursor protein, thereby generating abiologically active hG-CSF protein consisting of 174 amino acids (Nagataet al., EMBO J. 5:575-581, 1986; Hill et al., Proc. Natl. Acad. Sci. USA90:5167-5171, 1993).

The present inventors discovered, during their research into cloning ofhG-CSF gene and production of its translation product, that G-CSF cDNAderived from tumor cells has a deletion of the entire exon 3 region (108bp). Although deletion of specific exons has been reported in a varietyof genes not including G-CSF gene, such deletion in hG-CSF gene has beenunknown until now. The hG-CSF protein is known to be present in a formhaving or not having three amino acids corresponding to the 3′ end ofexon 2, while being biologically active in both of the two forms (Nagataet al., EMBO J. 5:575-581, 1986). Therefore, a method of diagnosingcancer based deletion of exon 3 of G-CSF gene may be applied to allsubtypes of G-CSF according to an identical principle. In addition,since other mammalian-derived G-CSF proteins are known to have almostidentical biological activity to the hG-CSF protein, it will beunderstood by those skilled in the art that a method of diagnosingcancer based on detection of deletion of exon 3 of G-CSF gene accordingto the present invention can be applied to other mammalian-derived G-CSFproteins according to an identical principle.

Both genes specifically expressed or suppressed in tumor cells, andgenetic mutation, can be detected by the conventional molecularbiological methods, which are exemplified by (a) polymerase chainreaction (PCR) (Bottema, C. D., Mutat Res. 233:93-102, 1993; Nelson, D.L., Curr. Opin. Genet. Dev. 1: 62-68, 1991; Pourzand, C. and Cerutti,P., Mutat. Res. 288:113-121, 1993; and Holland P M et al., Proc. Natl.Acad, Sci. USA 8: 7276-7280, 1991), (b) single-stranded conformationpolymorphism (SSCP) (Glavac D., Hum. Mutat. 19:384-394, 2002; Strippoli,P. et al., Int. J. Mol. Med 8:567-572, 2001; and Methods Mol Biol187:151-63, 2002), (c) DNA sequencing analysis (Sanger, F. et al., Proc.Natl. Acad. Sci USA 74:5463-5467, 1997), (d) protein truncation test(PTT) (Hardy, C. A., Methods Mol. Biol. 187:87-108, 2002), (e) automaticnucleotide sequence analysis (Boutin P. et al., Hum. Mutat.15(2):201-203, 2000), (f) study of loss of heterozygosity (LOH) (Yang Q.et al., Clin. Cancer Res. 8:2890-2893, 2002), (g) study ofmicrosatellite instability (MSI) (Furlan, D. et al., J Pathol197:603-609, 2002), (h) gene analysis using MALDI-TOF (Leushner J.,Expert Rev. Mol. Diagn. 1:11-18, 2001), (i) gene analysis byhybridization (Wetmur, J. G., Critical Reviews in Biochem. Mol. Biol.26:227-259, 1991), (j) gene analysis using DNA chips (Goessl et al.,Urology 58:335-338, 2001; Zhou et al., Breast Cancer Res. Treat.66:217-224, 2001; and C K Kim et al., Korean Pat Publication No.2001-0061173), and (k) analysis using protein chips (Pharmacogenomics1:385-393, 2000). It will be understood by those skilled in the art thatdeletion of the exon 3 region of G-CSF gene or protein can be easilydetected by using the conventional molecular biological methodsincluding the examples as described above. The preferred molecularbiological methods used in detecting deletion of the exon 3 region ofG-CSF gene or protein include PCR, hybridization, DNA chips, proteinchips and enzyme-linked immunosorbent assay (ELISA).

To perform cancer diagnosis according to the present invention, a G-CSFgene or protein sample should first be obtained from tissue specimens orcells. Since a DNA sample for a specific gene is typically obtained fromnormal tissues or cells at a very small amount, the specific gene shouldbe amplified by PCR, and, for such amplification, suitable primersshould be designed. In the present invention, to amplify a part or anentire region of exon 3 of G-CSF gene, nucleic acid fragments to be usedas primers in PCR for detection of deletion of exon 3 are necessary.That is, the primers, as used herein, refer to oligonucleotides capableof amplifying a nucleotide sequence of G-CSF gene, comprising a part oran entire region of exon 3. Those skilled in the art will be able toeasily design such primers. Therefore, all primers capable of amplifyingG-CSF gene comprising a part or an entire region of exon 3, which can bedesigned by those skilled in the art, are intended to fall within thescope of the present invention. Examples of the primers includeoligonucleotides designated as SEQ ID NOs. 1 and 2, which are capable ofamplifying a region (Thr1-Pro174) ranging from a part of exon 2 to exon5 of hG-CSF gene, oligonucleotides designated as SEQ ID NOs. 3 and 5,which are capable of amplifying a region (Ile24-Leu71) ranging from apart of exon 2 to exon 3 of hG-CSF gene, and oligonucleotides designatedas SEQ ID NOs. 4 and 6, which are capable of amplifying a region(Cys36-Ser80) ranging from exon 3 to a part of exon 4 of hG-CSF gene.The present inventors investigated the presence or absence of G-CSF geneand exon 3 in the G-CSF gene in mRNA and cDNA samples obtained from 8normal tissues and 17 tumor cell lines.

In accordance with the present invention, there are provided nucleicacid fragments for use as primer sets in detecting deletion of exon 3through amplification of the exon 3 region of G-CSF gene, which include,but are not limited to, the following sets, each of which consists of asense primer and an antisense primer:

(SEQ ID NO. 1) sense: 5′-ACCCCCCTGGGCCCTGCC-3′ and (SEQ ID NO. 2)antisense: 5′-TCAGGGCTGGGCAAGGTG-3′; (SEQ ID NO. 1) sense:5′-ACCCCCCTGGGCCCTGCC-3′ and (SEQ ID NO. 5) antisense:5′-CAGCTGCAGGGCCTGGCT-3′; (SEQ ID NO. 1) sense: 5′-ACCCCCCTGGGCCCTGCC-3′and (SEQ ID NO. 6) antisense: 5′-CGCTATGGAGTTGGCTCAAGC-3′;(SEQ ID NO. 1) sense: 5′-ACCCCCCTGGGCCCTGCC-3′ and (SEQ ID NO. 9)antisense: 5′-CAGCTTCTCCTGGAGCGC-3′; (SEQ ID NO. 3) sense:5′-ATCCAGGGCGATGGCGCAGCG-3′ and (SEQ ID NO. 2) antisense:5′-TCAGGGCTGGGCAAGGTG-3′; (SEQ ID NO. 3) sense:5′-ATCCAGGGCGATGGCGCAGCG-3′ and (SEQ ID NO. 5) antisense:5′-CAGCTGCAGGGCCTGGCT-3′; (SEQ ID NO. 3) sense:5′-ATCCAGGGCGATGGCGCAGCG-3′ and (SEQ ID NO. 6) antisense:5′-CGCTATGGAGTTGGCTCAAGC-3′; (SEQ ID NO. 4) sense:5′-TGTGCCACCTACAAGCTGTGC-3′ and (SEQ ID NO. 2) antisense:5′-TCAGGGCTGGGCAAGGTG-3′; (SEQ ID NO. 4) sense:5′-TGTGCCACCTACAAGCTGTGC-3′ and (SEQ ID NO. 5) antisense:5′-CAGCTGCAGGGCCTGGCT-3′;  (SEQ ID NO. 4) sense:5′-TGTGCCACCTACAAGCTGTGC-3′ and (SEQ ID NO. 6) antisense:5′-CGCTATGGAGTTGGCTCAAGC-3′.

The nucleic acid fragments include oligonucleotides capable of detectingdeletion of exon 3 of G-CSF gene in spite of not containing a nucleotidesequence corresponding to the exon 3 region, wherein theoligonucleotides may contain a nucleotide sequence corresponding to exon2 or exon 4 of G-CSF gene.

In accordance with an aspect of the present invention, there is provideda gene microarray or membrane to which a DNA fragment comprising a partor an entire region of exon 3 of the G-CSF gene is immobilized, which isuseful for diagnosis of cancer. The gene microarray includes DNA chipseffective for detection of a gene corresponding to a probe byhybridization including applying an oligonucleotide probe on the surfaceof a slide glass treated with a specific chemical reagent. Non-limitingexamples of the membrane, which can be used instead of the slide glassin hybridization, include all membranes capable of immobilizing DNAfragments, and preferably, nylon and nitrocellulose membranes.

A nucleic acid fragment corresponding to exon 3 of the G-CSF gene isattached on the surface of the microarray according to the presentinvention, along with one or more nucleic acid fragments selected fromthe group consisting of nucleic acid fragments corresponding to exons 1,2, 4 and 5 of the G-CSF gene, wherein each of the nucleic acid fragmentsused as probes may contain a part or an entire region of itscorresponding exon. Non-limiting examples of the nucleic acid fragmentcorresponding to the exon 3 region, used as a probe in the presentinvention, include oligonucleotides having a nucleotide sequencedesignated as SEQ ID NO. 14: TGTGCCACCTACAAGCTGTG, a nucleotide sequencedesignated as SEQ ID NO. 15: GAGCTGGTGCTGCTCGGACA, a nucleotide sequencedesignated as SEQ ID NO. 16: GGACACTCTCTGGGCATCCC, and a nucleotidesequence designated as SEQ ID NO. 17: CTGAGCAGCTGCCCCAGCCA. Non-limitingexamples of the nucleic acid fragments corresponding to exons 1, 2, 4and/or 5, which are used as control probes, include oligonucleotideshaving a nucleotide sequence designated as SEQ ID NO. 10:CTGCAGCTGCTGCTGTGGCAC, a nucleotide sequence designated as SEQ ID NO.12: AGAAGCTGTGGTGCCAC, a nucleotide sequence designated as SEQ ID NO.13: TGAGTGAGTGTGCCAC, a nucleotide sequence designated as SEQ ID NO. 18:GCAGGC TGCTTGAGCCAA, a nucleotide sequence designated as SEQ ID NO. 19:AGAAGCTGGCAGGCTG, and a nucleotide sequence designated as SEQ ID NO. 20:TGAGTGAGGCAGGCTG.

Spotting the probes on the surface of a slide glass and a membrane canbe easily achieved by the conventional technique known in the art. Inaddition, preparation of probes, hybridization and stripping will beperformed according to the conventional techniques common in the art.

In another aspect of the present invention, there is included acomposition for diagnosis of cancer, comprising a DNA fragmentcontaining a part or an entire region of the exon 3 region of G-CSF geneand a diagnostically acceptable carrier. In a further aspect of thepresent invention, there is included a method of diagnosing canceremploying a mutated G-CSF protein having a deletion of an amino acidsequence corresponding to the exon 3 region of the G-CSF gene, and apolyclonal or monoclonal antibody to the mutated G-CSF protein. The term“a mutated G-CSF protein having a deletion of an amino acid sequencecorresponding to the exon 3 region of G-CSF gene”, as used herein,refers to a mutated G-CSF protein produced by a deletion in a regionranging from exons 1 to 5 during expression of G-CSF gene, essentiallycontaining a deletion in exon 3. In a still further aspect of thepresent invention, there is included a diagnostic kit comprising a DNAfragment containing a part or an entire region of exon 3 of the G-CSFgene and a DNA microarray using the DNA fragment. In a still furtheraspect of the present invention, there is included a diagnostic kitcomprising a mutated G-CSF protein having a deletion of an amino acidsequence corresponding to the exon 3 region of the G-CSF gene, and aprotein microarray using a polyclonal or monoclonal antibody to themutated G-CSF protein.

The mutated G-CSF protein having a deletion of an amino acid sequencecorresponding to the exon 3 region of the G-CSF gene, and the polyclonalor monoclonal antibody to the mutated G-CSF protein, according to thepresent invention, may be produced by the conventional method common inthe art (Harlow, E. and Lane, D., Antibodies. A Laboratory Manual. ColdSpring Harbor, N.Y.: Cold Spring Harbor Laboratory, 1988; and Wilson, L.and Matsudaira, P. eds. Antibodies in Cell Biology (Methods in CellBiology, Vol. 37). New York: Academic Press, 1933).

In an embodiment of the present invention, when amplifying the exon 3region of G-CSF gene in mRNA and cDNA samples obtained from 17 tumorcell lines and analyzing the nucleotide sequence of the products, theexon 3 region (108 bp) was found to be deleted in hG-CSF cDNA derivedfrom various tumor cell lines, including stomach cancer cells, breastcancer cells, sarcoma cells, intestinal cancer cells, lung cancer cells,cervical cancer cells and malignant melanoma cells (see, FIGS. 2 and 3).When performing PCR using the hG-CSF cDNA derived from the 17 tumor celllines as a template with a primer set according to the presentinvention, it was found that PCR products derived from 16 tumor celllines are smaller in size than those from a normal cell line, and, asidentified by nucleotide sequence analysis of the PCR products, have adeletion of the exon 3 region, among five exons of the normal G-CSFgene.

The deletion of the exon 3 region in G-CSF cDNA can be detected byhybridization. For example, after obtaining a DNA fragment correspondingto exon 3 and a DNA fragment corresponding to exon 2 by performing PCRusing normal hG-CSF gene as a template, and immobilizing the two DNAfragments on a nylon membrane, the nylon membrane is hybridized with acDNA target, derived from a tumor cell line, and deletion of the exon 3region is determined by detecting binding of the probe with the exon 3DNA fragment immobilized on the membrane.

The deletion of the exon 3 region in G-CSF cDNA can be detected using aDNA chip, by immobilizing oligonucleotides corresponding to each exon ofthe G-CSF gene on a slide glass, preparing probes by PCR using exons ofthe hG-CSF gene as templates, and then reacting the probes with theoligonucleoties.

In addition, deletion of the exon 3 region of G-CSF may be easilydetected by preparing a recombinant G-CSF protein using a mutated G-CSFnucleotide sequence having a deletion of the exon 3 region, preparing apolyclonal or monoclonal antibody to the recombinant mutated G-CSFprotein, and comparing the mutated G-CSF protein levels in normalindividuals and cancer patients by ELISA using the antibody.

In another aspect, the present invention includes animmunochromatographic assay. The representative example of the assay isthe lateral flow immunoassay. A diagnostic kit for lateral flowimmunoassay comprises a sample pad to which a specimen is loaded, areleasing pad coated with an antibody as a probe, a membrane (mainly,nitrocellulose) or strip for development of the sample, in which thespecimen migrates and is separated and antibody-antigen reaction occurs,and an absorption pad for driving continuous migration of the specimen.The antibody used as a probe is labeled by being immobilized on, forexample, colloidal gold particles. Instead of the colloidal goldparticles, latex beads or carbon particles are available. The diagnostickit for lateral flow immunoassay is typically designed to detect ananalyte in a sandwich form. The analyte contained in the specimen isapplied to the sample pad, migrates and reacts with the antibody coatedon the releasing pad, forming antigen-antibody complexes. The formedcomplexes further migrate and are captured by an additional antibodyimmobilized on the membrane for development, generating triplexes ofsandwich form. That is, since the additional antibody is immobilized onthe membrane for development, the triplexes are accumulated on thesurface of the membrane, on which the additional antibody isimmobilized. Since proteins are not visible to the naked eye, formationof triplexes and their amount are determined by amount of gold particlesconjugated to the antibody contained in the triplexes.

In accordance with another aspect of the present invention, there isprovided a method of diagnosing cancer based on detection of deletion ofthe exon 3 region of G-CSF gene. In detail, in accordance with thepresent invention, diagnosis of cancer may be achieved by obtaining anucleic acid sample from animal tissues or cells, and detecting deletionof the exon 3 region of G-CSF gene in the nucleic acid sample using amolecular method. Human tissues or cells as a source of the nucleic acidsample include biological fluid samples, biopsy specimens, solid phasetissue samples such as tissue culture or the tissue-derived cells, andoffsprings of the cells. In addition, the sample sources includechemical reagent-treated, solubilized samples, cultured cells, cellculture supernants, and cell lysates. In more detail, in terms ofobjects of the present invention, the human tissues or cells includetumor tissues or tissues considered to be neoplastic, and may beobtained by conventional methods common in the art, such as surgicalresection, biopsies or suction. From the human tissues or cells, anucleic acid sample may be obtained by the conventional method known inthe art.

The method of diagnosing cancer based on detection of deletion of exon 3of G-CSF gene according to the present invention may be, as describedabove, achieved by nucleotide sequence analysis of the G-CSF gene, aswell as by using a specific probe to a region corresponding to the exon3 region of the G-CSF gene, for example, by employing an antibodyspecific to a mutated G-CSF protein having a deletion of an amino acidsequence corresponding to the exon 3 region.

The method of diagnosing cancer of the present invention may be used indiagnosis of various cancers, including stomach cancer, breast cancer,sarcoma, intestinal cancer, lung cancer, cervical cancer, liver cancer,prostate cancer, tongue cancer, laryngeal cancer, pharyngeal cancer,oral cancer, thyroid cancer, colorectal cancer, esophageal cancer, andtesticular cancer.

The present invention will be explained in more detail with reference tothe following examples in conjunction with the accompanying drawings.However, the following examples are provided only to illustrate thepresent invention, and the present invention is not limited to them.

Example 1 Preparation of mRNA and cDNA from Tumor Cell Lines

mRNA and cDNA samples were prepared from 8 normal cell lines andtissues, and 17 tumor cell lines. The normal cell lines and tumor celllines used in Examples of the present invention are given in Table 1,below.

TABLE 1 Normal and tumor cell lines used in the present invention Celltypes Cell collection centers Tumor cell YCC-7 Stomach cancer cell lineCancer metastasis research center, College of Medicine, lines YonseiUniversity AGS Stomach cancer cell line ATCC CRL-1739 SNU-1 Stomachcancer cell line Korean Cell Line Research Foundation (KCLRF), SeoulNational University MDA-MB-231 Breast cancer cell line ATCC HTB-26 MCF-7Breast cancer cell line ATCC HTB-22 SK-BR-3 Breast cancer cell line ATCCHTB-30 HT-1080 Sarcoma cell line ATCC CCL-121 HCT-116 Colon cancer cellline ATCC CCL-247 COLO205 Colon cancer cell line ATCC CCL-222 DLD-1Colon cancer cell line ATCC CCL-221 HT-29 Colon cancer cell line ATCCHTB-38 A549 Lung cancer cell line ATCC CCL-185 NCI-H460 Lung cancer cellline ATCC HTB-177 HeLa Cervical cancer cell line ATCC CCL-2 C-33ACervical cancer cell line ATCC HTB-31 B16 Malignant melanoma cell lineATCC CRL-6322 U-87MG Brain cancer cell line ATCC HTB-14 Normal cell 293Human kidney embryonic Cancer metastasis research center, College ofMedicine, lines cell line Yonsei University Sample 1 Human lymphocytesCancer metastasis research center, College of Medicine, YonseiUniversity Sample 2 Human monocytes Cancer metastasis research center,College of Medicine, Yonsei University Sample 3 Human epidermis tissuesCancer metastasis research center, College of Medicine, YonseiUniversity Sample 4 Human dermis tissues Cancer metastasis researchcenter, College of Medicine, Yonsei University Sample 5 Human hairpollicle cells Cancer metastasis research center, College of Medicine,Yonsei University Sample 6 Human fat cells Cancer metastasis researchcenter, College of Medicine, Yonsei University Sample 7 Human musclecells Cancer metastasis research center, College of Medicine, YonseiUniversity

The tumor cell lines listed in Table 1 can be obtained from the cellcollection centers listed in Table 1. In addition, human normal celllines, lymphocytes, monocytes, epidermis, dermis, hair pollicles, fatcells and muscle cells can be easily obtained from the cancer metastasisresearch center at College of Medicine, Yonsei University. The tumorcell line YCC-7, obtained from the cancer metastasis research center,was prepared as follows. Ascitic fluid was aseptically obtained fromadvanced cancer patients, and supplemented with heparin in an amount of10 units per ml to prevent clumping of cells. After centrifugation at400×g for 10 min, the precipitated cells were cultured in a cultureflask of 25 cm³. In case of containing a large number of erythrocytes,Ficoll-hypaque density gradient centrifugation at 800×g was performed toseparate mononuclear cells from erythrocytes, and the obtainedmononuclear cell phase was incubated at 37° C. under 5% CO₂. Afterincubation for 16-18 hrs, the culture medium was centrifuged at 400×gfor 10 min, and the precipitated cells were cultured in a new cultureflask of 25 cm³. During culturing, cells were observed under a phasecontrast microscope, and the culture medium was replaced twice or threetimes per week. When tumor cell colonies were formed, the tumor cellclusters were obtained by treatment with trypsin-EDTA or by usingscrapers, and the fluid containing tumor cells was centrifuged to removenormal cells. The resulting pure tumor cells were stored at frozenstates according to their passages.

Total RNA was isolated from each tumor cell line, normal cell line andnormal tissue using Tri-Reagent (Gibco-BRL, USA). 1 ml of Trizol Reagentwas added to a tissue sample ground after quickly freezing using liquidnitrogen, followed by incubation at room temperature for 5 min. Theresulting tissue sample was supplemented with 0.2 ml of chloroform,vigorously mixed for 15 sec, and incubated at room temperature for 5min. After centrifugation at 12,000×g at 4° C. for 15 min, the resultantaqueous phase was transferred to a new tube. An equal volume ofisopropanol was added to the tube, and the tube was placed at 4° C. for10 min. After centrifugation at 12,000×g at 4° C. for 10 min, thesupernatant was carefully discarded, and the pellet was washed with 70%ethanol, followed by centrifugation at 7,500×g at 4° C. for 5 min. Afterbeing dried, the RNA pellet was dissolved in RNase-free water.

To synthesize cDNA from mRNA isolated from each cell line, andhuman-derived tumor and normal cell line, RT-PCR was performed asfollows. 2 μg of total RNA was mixed with 1 μl of an oligo(dT)₁₆-primer,and RNase-free water was added up to a final volume of 11 μl. Thismixture was heated at 90° C. for 5 min, and placed on ice, immediatelyafter completion of the heating. After putting 4 μl of a reactionbuffer, 2 μl of 10 mM dNTPs, 1 μl of RNase inhibitor and 2 μl of reversetranscriptase into another tube, 8.5 μl of the RNA mixture was added tothe pre-mixture tube, followed by incubation at room temperature for 10min. The reaction mixture was incubated at 42° C. for 90 min, and thenat 95° C. for 15 min. Immediately after the incubation at 95° C., themixture was placed on ice to terminate reaction, thus yielding a cDNAsample.

Example 2 Detection of hG-CSF Gene by PCR

In order to detect expression of normal hG-CSF gene in each tumor cellline, PCR was carried out using cDNA prepared in Example 1 as atemplate. As shown in FIG. 2, PCR reactions were divided into threetypes according to their amplified products, as follows: PCR 1 foramplification of a region (Thr1-Pro174) ranging from a part of exon 2 toexon 5 of hG-CSF gene; PCR 2 for amplification of a region (Ile24-Leu71)ranging from a part of exon 2 to exon 3 of hG-CSF gene; and PCR 3 foramplification of a region (Cys36-Ser80) ranging from exon 3 to a part ofexon 4 of hG-CSF gene.

PCR 1 was carried out using a cDNA sample from each tumor cell line as atemplate, and a primer set of a sense primer designated SEQ ID NO.: 1(5′-ACCCCCCTGGGCCCTGCC-3′) and an antisense primer designated SEQ IDNO.: 2 (5′-TCAGGGCTGGGCAAGGTG-3′). PCR 2 was carried out using a cDNAsample from each tumor cell line as a template, and a primer set of asense primer designated SEQ ID NO.: 3 (5′-ATCCAGGGCGATGGCGCAGCG-3′) andan antisense primer designated SEQ ID NO.: 5 (5′-CAGCTGCAGGGCCTGGCT-3′).PCR 3 was carried out using a cDNA sample from each tumor cell line as atemplate, and a primer set of a sense primer designated SEQ ID NO.: 4(5′-TGTGCCACCTACAAGCTGTGC-3′) and an antisense primer designated SEQ IDNO.: 6 (5′-CGCTATGGAGTTGGCTCAAGC-3′). PCR was performed using a highfidelity PCR system (Boehringer Mannheim Co., Germany) under thefollowing condition. PCR conditions included denaturation at 94° C. for7 min, and 30 cycles of denaturation at 94° C. for 40 sec, annealing at56° C. for 40 sec and extension at 72° C. for 1 min, followed by finalextension at 72° C. for 7 min.

PCR products were separated on an agarose gel. As a result ofelectrophoresis, in the case of PCR1, PCR products from tumor cell lineswere found to be smaller in size than a normal G-CSF gene, except that aPCR product from U-87MG has a size equal to that of normal G-CSF gene(see, FIG. 3A in which M: size marker, lane 1: normal cell line, lane 2:YCC-7, lane 3: AGS, lane 4: SNU-1, lane 5: MDA-MB-231, lane 6: MCF-7,lane 7: SK-BR-3, lane 8: HT-1080, lane 9: HCT-116, and lane 10: COLO205;and FIG. 3B in which M: size marker, lane 1: normal cell line, lane 2:DLD-1, lane 3: HT-29, lane 4: A549, lane 5: NCI-H460, lane 6: HeLa, lane7: C-33A, lane 8: B16, and lane 9: U-87MG). In the cases of PCR2 andPCR3, a PCR product was generated only from U-87MG cells, while PCRproducts were not obtained in other tumor cell lines (see, FIGS. 6 and7). In addition, the PCR product of U-87MG cells, produced in PCR2 andPCR3, was found to have a size equal to that of the PCR product ofnormal cells.

Nucleotide sequences of PCR1 products were analyzed using an automaticDNA sequencer (ABI Prism model 377, Perkin Elmer Co., USA). As a resultof nucleotide sequence analysis, the PCR product obtained from U-87MGcells was found to have a nucleotide sequence (SEQ ID NO.: 7) identicalto that of G-CSF gene of normal cells. In contrast, PCR productsobtained from other tumor cell lines, compared with the nucleotidesequence of G-CSF of normal cells, was found to have a nucleotidesequence (SEQ ID NO.: 8) having a deletion of 108 by (see, FIGS. 4 and5). When comparing the deleted 108 bp with the nucleotide sequence ofG-CSF, the deleted 108 bp was found to correspond to the exon 3 regionamong five exons.

In order to investigate whether tissues of normal individuals displaythe same PCR result as obtained from the normal cell line, when RT-PCRwas carried out using RNA isolated from lymphocytes, monocytes,epidermis tissues, dermis tissues, hair pollicles, fat cells and musclecells of normal individuals, and PCR was carried out using cDNA obtainedfrom the RT-PCR, no deletion of exon 3 was found in cells and tissuesfrom normal individuals (see, FIG. 11), indicating that the deletion ofexon 3 found in PCR products of tumor cell lines is not induced by PCRerror. In addition, a mutated G-CSF protein expressed from G-CSF cDNAhaving a deletion of exon 3 is highly likely to have lost active sitefunction and to have a different conformation from the normal G-CSFprotein. Therefore, it is believed that G-CSF protein expressed in tumorcells does not exhibit normal function.

Example 3 Detection of G-CSF Gene by Hybridization

The deletion of exon 3 in G-CSF cDNA from tumor cell lines was detectedby hybridization.

PCR was carried out using G-CSF gene derived from a normal cell line asa template, and a primer set of a sense primer designated SEQ ID NO.: 4(5′-TGTGCCACCTACAAGCTGTGC-3′) and an antisense primer designated SEQ IDNO.: 5 (5′-CAGCTGCAGGGCCTGGCT-3′). A DNA fragment of 108 bycorresponding to the exon 3 region of G-CSF gene was obtained.

Separately, PCR was carried out using G-CSF gene derived from the normalcell line as a template, and a primer set of a sense primer designatedSEQ ID NO.: 1 (5′-ACCCCCCTGGGCCCTGCC-3′) and an antisense primerdesignated SEQ ID NO.: 9 (5′-CAGCTTCTCCTGGAGCGC-3′). A DNA fragment of105 by corresponding to the exon 2 region of G-CSF gene was obtained.

After being purified, each of the DNA fragments (50 ng/μl) was spottedon a nylon membrane (Boehringer Mannheim, Germany), and incubated at 80°C. for 2 hrs to immobilize it onto the membrane.

Target probes for hybridization of the DNA fragments corresponding toexon 2 and exon 3, immobilized on the membrane, were prepared by RT-PCR.RT-PCR was performed as follows. First, a reaction mixture A (2 μg oftotal RNA, 1 μl of an oligo(dT)₁₆-primer, total volume: 15 μl) wasincubated at 94° C. for 2 min to denature RNA, and then slowly cooled to42° C. over about 20 min. Another reaction mixture B (333 μM of each ofdATP, dGTP and dCTP, 1× reverse transcriptase buffer, 20 μCi of [α-³²P]dCTP (2,000-3,000 Ci/mmol), 50 U of AMV reverse transcriptase, totalvolume: 30 μl) was added to the mixture A, and reverse transcription wascarried out at 42° C. for 2 hrs. Thereafter, dNTP, including [α-³²P]dCTP, not participating in polymerization were removed using a QIAquickNucleotide Removal Kit (Qiagen, USA).

Using the cDNA probe, hybridization was carried out on the nylonmembranes on which the DNA fragments corresponding to exons 2 and 3 ofG-CSF are immobilised. First, after being immersed in 2×SSPE buffer(1×SSPE: 0.18M NaCl, 10 mM sodium phosphate, 1 mM EDTA, pH 7.7) for 5min, the nylon membranes were treated with 2 ml of a hybridizationsolution (5×SSPE, 2% SDS, 1×Denhardt's reagent, sonicated and 100 μg/mldenatured salmon sperm DNA) preheated to 65° C., where the membraneswere sealed in a vinyl bag. After incubation at 65° C. for 1 hr, adenatured cDNA probe, prepared by mixing 10 μl of the cDNA probe with0.5 ml of the hybridization solution and then boiling the mixture for 10min, was added to the hybridization solution in which the membranes wereimmersed, followed by incubation at 65° C. for 18 hrs to allowhybridization. Thereafter, the membranes were washed three times with awashing solution (0.5×SSPE, 0.2% SDS) at 65° C. for 30 min.Radioactivity was detected by exposing the membranes or X-ray film anddeveloping the film.

As shown in FIG. 8, in the case of probes prepared using total RNA fromtumor cell lines, YCC-7, AGS, HT-29, A549 and MCF-7, the probes werefound to bind to the DNA fragment of exon 2, but not to bind to the DNAfragment of exon 3 (see, FIGS. 8A, 8B, 8C, 8D and 8E). In contrast, aprobe prepared using total RNA from U-87MG cells known to contain normalG-CSF gene was found to bind the two DNA fragments of exons 2 and 3(see, FIG. 8F). As described above, it was demonstrated that deletion ofexon 3 of G-CSF can be easily detected by hybridization. Thehybridization method is applicable to detection methods using therecently developed DNA microarray. Therefore, it is believed that avariety of detection methods using the hybridization method can beeasily developed, and that cancer can be diagnosed easily and accuratelyby the detection methods.

Example 4 Detection of G-CSF Gene Using a DNA Chip for Detection ofDeletion of Exon 3 of G-CSF

In order to investigate whether a DNA chip can be used as a tool fordetection of deletion of exon 3 of G-CSF mRNA or cDNA, various DNAfragment probes capable of being immobilized on a glass plate wasprepared as follows.

One probe corresponding to a part of exon 2 of G-CSF, fournon-overlapping probes corresponding to exon 3, and one probecorresponding to a part of exon 4, were designed to consist of 20nucleotides each. In addition, one probe corresponding to a regioncontinuously ranging from a part of exon 2 to a part of exon 3, oneprobe corresponding to a region continuously ranging from a part of exon3 to a part of exon 4, and one probe corresponding to a regioncontinuously ranging from a part of exon 2 to a part of exon 4, weredesigned to have 8 nucleotides of each exon. Since two different G-CSFmRNAs (human G-CSFa and human G-CSFb mRNAs) are generated by alternativesplicing in the exon 2 region (Tshuchiya M. et al., EMBO J 5:575-581,1986), two types of probes comprising a region corresponding to exon 2were prepared, based on the two different G-CSF mRNAs.

To confer ability to be immobilised on a glass plate, when synthesizingall DNA fragment probes, a base having an amino group was inserted tothe 3′ end of each of the probes using an aminolinker column (Cruachem,Glasgrow, Scotland), and slide glass coated with aldehyde residues (CELAssociates, Inc., Houston, Tex., USA) were used. After being dissolvedin 3×SSC (0.45M NaCl, 15 mM C₆H₅Na₃O₇, pH 7.0), the DNA probes wereimmobilised on the slide glass by accumulating the DNA probes using amicroarrayer manufactured by the present inventors (Yoon et al, J.Microbiol. Biotechnol. 10:21-26, 2000), and reacting for over 1 hr underabout 55% humidity, and then leaving the glass at room temperature for 6hrs. Herein, the probes were arranged at intervals of 275 μm on theglass at an amount of 100 μM, thus producing a microarray.

Immobilization of probes through reaction between amine groups of probesand aldehyde groups on the glasses was estimated by staining with SYBROgreen II (Molecular Probe, Inc., Leiden, Netherlands).

A gene fragment as a probe, to be immobilized on a glass, was preparedby asymmetric PCR using G-CSF gene extracted from each cell line, and aprimer set of a sense primer designated SEQ ID NO.:10(5′-CTGCAGCTGCTGCTGTGGCAC-3′) and an antisense primer designated SEQ IDNO.:11 (5′-FITC-CTGCTGCCAGATGGTGGT-3′) in a ratio of 1:5, wherein thegene fragment was obtained by performing PCR once. Information on probesimmobilized on the slide glass is given in Table 2, below.

TABLE 2 Nucleotide Corresponding regions SEQ sequences on hG-CSF geneID NOs. CTGCAGCTGCTGCTGTGGCAC Exon 2 10 AGAAGCTGTGGTGCCAC Exons 2 to 312 (hG-CSFa) TGAGTGAGTGTGCCAC Exons 2 to 3 13 (hG-CSFb)TGTGCCACCTACAAGCTGTG Exon 3 14 GAGCTGGTGCTGCTCGGACA Exon 3 15GGACACTCTCTGGGCATCCC Exon 3 16 CTGAGCAGCTGCCCCAGCCA Exon 3 17GCAGGCTGCTTGAGCCAA Exon 4 18 AGAAGCTGGCAGGCTG Exons 2 to 4 19 (hG-CSFa)TGAGTGAGGCAGGCTG Exons 2 to 4 20 (hG-CSFb)

Asymmetric PCR was carried out under the conditions of denaturation at94° C. for 5 min, 10 cycles of denaturation at 94° C. for 1 min,annealing at 56° C. for 1 min and extension at 72° C. for 30 sec, and 30cycles of denaturation at 94° C. for 1 min, annealing at 58° C. for 1min and extension at 72° C. for 30 sec, followed by final extension at72° C. for 7 min.

PCR products were separated on an agarose gel. From the result ofelectrophoresis, double stranded DNA and single stranded DNA fragmentswere produced in each PCR sample. After amplifying G-CSF gene byasymmetric PCR using a plasmid carrying exon 3-deleted G-CSF gene andanother plasmid carrying G-CSF having no deletion of exon 3, the twoamplified products were analyzed using a DNA chip. A hybridizationsolution (6×SSPE, 20% (v/v) formamide) was added to 15 μl of theamplified product up to a final volume of 200 μl. The mixture wasapplied on a slide glass having an immobilised probe, and the glass wascovered with a probe-clip press-seal incubation chamber (Sigma Co., St.Louis, Mo.), followed by incubation in a shaking incubator at 30° C. for6 hrs to induce binding of the probe to the amplified product.Thereafter, the glass was washed over 5 min with 3×SSPE (0.45M NaCl, 15mM C₆H₅Na₃O₇, pH 7.0), 2×SSPE (0.3M NaCl, 10 mM C₆H₅Na₃O₇, pH 7.0), andthen 1×SSPE (0.15M NaCl, 5 mM C₆H₅Na₃O₇, pH 7.0), and scanned usingscanarray 5000 (GSI Lumonics Inc., Bedford, Mass.).

As shown in FIG. 9, in case of the plasmid having no deletion of exon 3in G-CSF gene, signals was detected for all probes. In contrast, in caseof the exon 3-deleted G-CSF-containing plasmid, signals were detected onexon 2 and exon 4 region, wherein the plasmid has a nucleotide sequencecorresponding to G-CSFa-type RNA.

These results indicate that DNA chips capable of detecting deletion ofthe exon 3 region of G-CSF mRNA or cDNA can be developed using the abovementioned primers and probes.

Example 5 Production of a Recombinant Mutated G-CSF Protein

To produce a mutated G-CSF protein in a large scale by recombinant E.coli, fed-batch fermentation of E. coli BL21(DE3) (Novagen Inc., USA)carrying a recombinant plasmid pED-CSF4BLIIE was performed. ThepED-CSF4BLIIE plasmid was prepared by cloning a nucleotide sequencecorresponding to a mutated G-CSF protein having a deletion of the exon 3region into a plasmid pET21c (Novagen Inc., USA), as follows. First, PCRwas carried out using human breast cancer cDNA library as a template,and a primer set of primer 1 (forward primer) containing an EcoRI site:5′-GCGAATTCATGGCTGGACCTGCCACCCAG-3′ and primer 2 (reverse primer)containing a BamHI site: 5′-GCGGATCCTTATTAGGGCTGGGCAAGGTGGCG-3′

A PCR product was treated with EcoRI and BamHI, and cloned into pUC19(Stratagene, USA), thus giving a plasmid p19CSF (see, FIG. 12). TheG-CSF gene cloned into pUC19 does not contain the exon 3 region.

Using the plasmid p19CSF as a template, PCR was carried out with aprimer set of primer 4 (forward primer):5′-GCGAATTCATATGACCCCCCTGGGCCCTGCCA-3′ and primer 2 (reverse primer):5′-GCGGATCCTTATTAGGGCTGGGCAAGGTGGCG-3′.

A PCR product was treated with NdeI and BamHI, and cloned into plasmidpET21c. To change a codon corresponding to the first amino acid to acodon optimal in the bacterial translation system, PCR was carried outagain using as the forward primer a primer:5′-GCGAATTCATATGACTCCGTTAGGTCCAGCCAGC-3′ instead of the primer 4:5′-GCGAATTCATATGACCCCCCTGGGCCCTGCCAGC-3′ the produced DNA fragment wastreated with NdeI and BamHI, and cloned into a plasmid pET21c, thusgiving a plasmid pED-CSF4BLIIE (see, FIG. 13). Nutrients and additionalsubstances, used for fed-batch fermentation, are given in Table 3,below.

TABLE 3 Culture medium Additionally supplied solution Nutrients Conc.(g/L) Substances Conc. (g/L) (NH₂)₂HPO₄ 3.0 Glucose 700.0 KH₂PO₄ 7.0MgSO₄•7H₂O 15.0 MgSO₄•7H₂O 1.0 Yeast extract 50.0 Citric acid 0.8 Yeastextract 2.0 Glucose 20.0 Trace metal solution 3 (ml)

E. coli BL21(DE3) cells transformed with the plasmid pED-CSF4BLIIE wereincubated in a 250 ml flask with agitation at 37° C. for 8 hrs. 200 mlof the culture was inoculated into 1.8 L of a culture medium containedin a 5 L fermentor (NBS fermentor), and cultured at 37° C., where pH ofthe medium was constantly maintained at 6.8. pH was maintained using a28% NH₄OH solution. Pure oxygen was optionally supplied. Supply ofadditional substances was controlled by a pH-stat. That is, when pHincreased to 6.88, 2-3 g of glucose, 0.3 g of yeast extract and 0.1 g ofMgSO₄.7H₂O were automatically added. During culturing, glucoseconcentration was maintained below 5 g per liter. When OD₆₀₀ reached 30,1 mM IPTG (isopropyl-β-D-thiogalactopyranoside) was added to the mediumto induce high growth of bacteria, thus producing a high concentrationbacteria culture having an OD₆₀₀ value of 90. Amount of produced mutatedG-CSF protein was evaluated by measuring intensity of a protein bandusing a densitometer (see, FIG. 10). Normal mature G-CSF protein has amolecular weight of about 18.7 kDa, and the mutated G-CSF proteintranslated from the exon 3-deleted G-CSF cDNA has a molecular weight ofabout 13 kDa.

Example 6 Preparation of a Polyclonal Antibody Specific to the MutatedG-CSF Protein

Using the mutated G-CSF protein obtained from the recombinant E. coli inExample 5 as an antigen, a polyclonal antibody specific to the mutatedG-CSF protein was prepared as follows. After emulsifying 400 μl of thepurified mutated hG-CSF protein dissolved in a phosphate buffer in aconcentration of 1 mg/ml with an equal volume of Freund's adjuvant(BRL), the emulsion was intramuscularly injected four times into rabbits(10 weeks old) in intervals of 11 days. 10 days after the fourthinjection, blood was collected from the immunized rabbits by heartpuncture. The collected blood was incubated at room temperature for 30min, and then at 4° C. overnight for complete blood clotting. Aftercentrifugation at 2,500 rpm for 30 min, the supernatant, that is, serumwas obtained. Ammonium sulfate was added to the serum up to a finalconcentration of 40% to precipitate proteins. After dialyzing overnightin a 10 mM phosphate buffer (pH 7.0), antibody was purified using a DEAEAffi-Gel Blue gel (Bio-Rad Inc.) (Smith, C. P., Jensen, D., Allen, T.and Kreger, M. (Eds.) Information Resources for Adjuvants and AntibodyProduction. U.S. Dept. of Agriculture, 1997; and Hanly W. C. et al.,ILAR Journal 37:93-118, 1995).

Example 7 Preparation of a Monoclonal Antibody Specific to the MutatedG-CSF Protein

After emulsifying 100 μl of the purified mutated hG-CSF protein (1mg/ml) with an equal volume of Freund's adjuvant (BRL), the resultingemulsion was intraperitoneally injected three times into BALB/c mice(6-8 weeks old) in intervals of 2 weeks. After the third injection, ananti-mutant G-CSF protein antibody was found to be produced. Afteranother 2 weeks, the mice were boosted with 100 μg of the purifiedmutated hG-CSF protein. 3 days after the boosting, splenocytes wereobtained from the immunized mice, and mixed with SP2/0-Ag14 myelomacells at a ratio of 10:1, and fusion was induced by adding a 50%polyethyleneglycol 1500 solution to the cell mixture, followed byincubation for 3 min. After centrifugation at 1,200 rpm for 8 min, thecell pellet was suspended in a HAT RPMI-1640 medium containing 10% fetalcalf serum (FCS) at a density of 3.5×10⁶ cells/ml. 0.1 ml of the cellsuspension was then put into each well of a 96-well plate, and incubatedin a 5% CO₂ incubator at 37° C. After 3 days, 0.1 ml of the HATRPMI-1640 medium containing 10% FCS was added to each well of the plate,and half of the medium was replaced with a new medium every fourth day(Amyx, H. L., JAVMA 191:1287-1289, 1987; Akerstrom, B. et al., J Immunol135:2589-2592, 1985; and Anon, Vet Health Inspectorate 6 pp. Rijswijk,The Netherlands, 1989).

After selective culturing in the HAT medium, the fused cells werescreened for production of an anti-mutant G-CSF protein antibody byELISA, as follows. First, the mutated hG-CSF protein used in theimmunization of mice was diluted in 0.01 M carbonate-bicarbonate buffer(pH 9.6) in a concentration of 0.1 μg/ml, and 50 μl of the resultingdilution was added to each well of the plate, followed by incubation at4° C. overnight to allow binding of the protein to the bottom of eachwell. The plate was washed four times with PBST (phosphate buffersaline, 137 mM NaCl, 2.7 mM KCl, 10 mM Na₂HPO₄, 2 mM KH₂PO₄, 0.15% Tween20), and blocked with albumin by incubation at 37° C. for 30 min.Thereafter, 50 μl of the cell culture supernant was added to each well,and the plate was incubated at room temperature for 2 hrs, followed bywashing four times with PBST. After diluting a biotin-conjugatedanti-mouse immunoglobulin antibody, as a secondary antibody, in 0.1%BSA-PBST in a concentration of 1 μg/ml, 50 μl of the dilution was addedto each well, followed at 37° C. for 1 hr. After washing four times withPBST, 50 μl of a 1:1000 dilution of streptavidin-horseradish peroxidasein 0.1% BSA-PBST was added to each well, and incubated at 37° C. for 30min, followed by washing four times with PBST. 50 μl of atetra-methylbenzidine (TMB) solution was added to each well, andincubated at room temperature, where the TMB was used as a substrate forperoxidase. After terminating the reaction with 2N sulfate, opticaldensity was measured at 450 nm using an ELISA reader. Cells obtainedfrom ELISA-positive wells were subcloned three times by limitingdilutions, in which the cells were diluted to 0.3 cell per well, toobtain hydridoma cells producing an anti-mutant hG-CSF proteinmonoclonal antibody. An anti-mutant hG-CSF protein monoclonal antibodywas obtained from the stable hybridoma cells (Amyx, H. L., JAVMA191:1287-1289, 1987; Akerstrom, B. et al., J Immunol 135:2589-2592,1985; and Anon, Vet Health Inspectorate 6 pp. Rijswijk, The Netherlands,1989).

Example 8 Detection of Mutated G-CSF Protein Levels by ELISA

After being diluted in 0.01 M carbonate-bicarbonate buffer (pH 9.6), theanti-rabbit and anti-mouse mutated hG-CSF protein antibodies were addedto each well of plates, along with ceruloplasmin, and the plates wereincubated at 4° C. overnight to allow attachment of the antibodies tothe bottom of each well. After being washed twice with PBST (0.15% Tween20), the plates were blocked with 0.1% albumin at 37° C. for 1 hr. Afterwashing twice with PBST, 50 μl of standard diluent buffer and each ofspecimens from normal individuals and cancer patients were added to theplates and carefully mixed, and the plates was incubated at 37° C. for 2hrs, followed by washing four times with PBST. After being diluted in 10mM phosphate buffer containing 0.15 M NaCl, 2.5 μg of peroxidaseconjugated anti-human immunoglobulin antibody as a secondary antibodywas added to each well, and the plates were incubated at roomtemperature for 30 min, followed by washing four times with PBST. 50 μlof a tetra-methylbenzidine (TMB) solution was added to each well, andincubated at room temperature under a dark condition, where the TMB wasused as a substrate for peroxidase. After terminating the reaction with2.5N sulfate, optical density was measured at 450 nm using an ELISAreader.

As shown in FIG. 11, mutated hG-CSF protein levels were found to be muchhigher in cancer patients than that in normal individuals. Whencomparing mutated hG-CSF protein levels of cancer patients to eachother, breast cancer patients showed the highest level of the mutatedhG-CSF protein.

As exemplified in detail in the above Examples, diagnosis of cancer maybe easily performed by detecting deletion of exon 3 of G-CSF by PCR orusing DNA chips, or by detecting a mutated G-CSF protein by ELISA. Theconventional cancer biomarkers are unable to detect all kinds of cancer,as follows. The known cancer biomarkers having low organ specificity,such as CEA, BFP, TPA and IAP, have low sensitivity, thus generatingfalse positive data. Also, the biomarkers having high organsspecificity, which are exemplified as AFP, PIVKA II, Esterase I, CA19-9,CA50, Span-1 antigen, CA15-3 and BCA 225, are useful only for targetorgans. The diagnostic cancer marker based on deletion of the exon 3region of G-CSF, discovered by the present inventors, may be used toeasily diagnose cancer by immunochemical methods and molecular methods.Development and use of such a cancer marker may facilitate earlydiscovery of cancer, thus largely contributing to effective treatment ofcancer.

INDUSTRIAL APPLICABILITY

As described hereinbefore, the present invention provides a method ofdiagnosing cancer based on detection of deletion of the exon 3 region ofG-CSF gene. The method of diagnosing cancer may be applied for diagnosisof a broad range of cancers, not only a specific cancer, and haseffectiveness in easily diagnosing cancer by molecular methods, such asPCR, hybridization or use of DNA chips, or relatively simpleimmunochemical assays such as ELISA. In addition, a DNA chip fordetection of deletion of the exon 3 region of G-CSF is advantageous interms of enabling diagnosis of all kinds of cancer, in a simpler,quicker and more accurate manner than the conventional methods fordiagnosis of cancer, and dealing with a large number of clinicalspecimens at one time, thereby leading to development and advance ofhuman medicine and improvement of the public good, as well as largelycontributing to development of DNA-chip technologies.

In addition, the mutated G-CSF protein having a deletion of an aminoacid sequence corresponding to the exon 3 region, and an antibody to themutated G-CSF protein, invented by the present inventors, may facilitatedistinction of normal individuals and cancer patients. This factindicates that detection of only G-CSF mutants allows accurate diagnosisof cancer and application of a large number of clinical specimens, thuslargely contributing to development of protein-chip technologies.

1-14. (canceled)
 15. Method for using an isolated G-CSF mRNA or cDNAfragment which is missing exon 3 as a diagnostic cancer marker.